Description Small heat shock proteins (sHsps) are ubiquitous molecular chaperones essential for cell survival by acting as the first line of defense against aberrant protein aggregation. sHsps exhibit great structural com- plexity, a consequence of their tendency to populate a dynamic ensemble of conformational and oligomeric states at equilibrium, whose variations are exquisitely regulated by known and unknown features within the N- and C-termini and various environmental factors. Presumably due to their structural dynamics and various con- formational states, sHsps rapidly bind and sequester denatured target proteins, in an ATP-independent man- ner, to maintain protein homeostasis and prevent protein aggregate accumulation during disease and cellular stress. The chaperone activity of sHsps is stunningly complex, as sHsps must interact with a large number of substrates through non-specific interactions under a variety of cellular conditions, leaving a large gap in our understanding of structure and function mechanisms. Models suggest the presence of multiple substrate bind- ing sites that may be accessible or become accessible to interact with denaturing substrates over a dynamic range of sHsp oligomeric conformational states. In some cases, small oligomers may represent an ?activated? form of the sHsp, although details of the function of individual oligomeric states remain unknown. Current mod- els suggest a role for the highly variable N-terminal sequence in both substrate binding and regulation of oli- gomer dynamics. Our rationale is that understanding mechanisms of chaperone activity and substrate selec- tivity will translate into better understanding of non-specific protein-protein interactions contributing to cellular homeostasis. Guided by preliminary data, we will address the complexity of sHsp structure and function, using a multi-faceted approach to understand mechanisms involved in chaperone function. In Aim 1, we will develop a strategy to synthesize N-terminal sequences of two mammalian sHsps, HspB1 and HspB5, followed by con- jugation to gold nanoparticles, resulting in multivalent chaperone mimetics. Using biophysical assays such as dynamic light scattering and gel analysis, we will identify chaperone activity of the sHsp N-terminal sequences to map features of substrate selectivity to these unconserved regions of HspB1 and HspB5. In Aim 2, we will explore the role of individual oligomerization states in contributing to sHsp chaperone activity and substrate selectivity. We will design and express fusion proteins, using glutathione s-transferase (GST) to create dimeric GST-HspB1 and GST-HspB5 or use coiled-coil motifs to create trimers,tetramers, pentamers, and hexamers of sHsps. We will identify the chaperone activity of each individual oligomeric state and compare this to the dy- namic native system. Overall, the proposed research is significant because it will contribute to our fundamental understanding of the complex and dynamic function of sHsps, while providing specific information about the chaperone activity and substrate selectivity of sHsp N-terminal sequences and individual oligomeric states. Ul- timately, these insights have the potential to advance our understanding of complex sHsp structure and fuction.